The mammalian and human bodies enclose specific biological markers in-vivo, which may indicate various pathologies in the living body. Early detection of such markers could mean early detection of pathologies in the body, which would lead to better treatment. Each specific in-vivo marker can bind to a specific biological particle such as an antibody, peptide and other binding agents.
These markers may be detected by sensing an optical change which occurs due to binding of a binding agent to a marker in-vivo, for example, detecting fluorescence at a given bandwidth, emitted from a binding agent bound to a marker in-vivo.
An additional method of detecting binding between molecules is “Fluorescence resonance energy transfer” (FRET). In FRET, a molecule in its excited state can transfer energy to a second molecule proximate to it, to excite the second molecule as well. The distance between the molecules should typically be below 10 nm, so as to ensure the occurrence of the FRET procedure. One common configuration comprises one molecule attached to a solid phase while the other molecule may be in suspension. For example, the molecules may be two binding agents, one suspended in a fluid and one attached onto a solid phase in contact with the fluid. When both binding agents bind to an in vivo marker, they become sufficiently close to one another so as to undergo a FRET process. The optical change which may be detected in FRET is an emission of fluorescence from the binding agent excited second.
However, it may be difficult to sense an optical change of the same sort as described above when the quantity or concentration of an in-vivo marker is not high. It may also be difficult to detect specific bindings within an in-vivo environment, due to high background noise in this environment, i.e., reflected illumination other than the optical change to be detected.
There is, therefore, a need for an in-vivo sensing device which will have the ability to sense signals from in-vivo markers indicating pathology at a high signal to noise ratio.
Other methods of detecting markers include inhibiting fluorescence of a probe, and then, under certain conditions, such as conformational change or cleavage following marker attachment to the probe, activating it. One such method used for detection of nucleic acid markers is by using molecular beacons. Molecular beacons are single-stranded oligonucleotide hybridization probes that form a loop structure. The loop contains a sequence complementary to a target sequence, which is part of the marker being detected. At one end of the loop is attached a fluorophore and at the other end is a quencher. The loop conformation of molecular beacons causes the fluorophore and the quencher to be in high proximity to each other such that no fluorescence is exhibited. However, when molecular beacons hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables exhibition of fluorescence. The fluorophore that is no longer proximate to the quencher can now exhibit fluorescence.
Another method is by using a probe comprising more than one fluorophore attached to a backbone, wherein the fluorophores are positioned in great proximity to one another. The backbone may be a substrate to a marker being detected, which may be an enzyme. If a marker is present, the enzymatic marker cleaves the substrate. Following enzymatic cleavage the fluorophores draw away from one another and may exhibit fluorescence.
The methods mentioned above for detecting markers are efficient when the tests are being done in-vitro, in the case of molecular beacons, or when done in-vivo when the markers are intracellular and the probes detect them within the cells. However, there is a need for an in-vivo probe detecting presence of markers which are excreted from the cells.